Limits...
FAK signaling is critical for ErbB-2/ErbB-3 receptor cooperation for oncogenic transformation and invasion.

Benlimame N, He Q, Jie S, Xiao D, Xu YJ, Loignon M, Schlaepfer DD, Alaoui-Jamali MA - J. Cell Biol. (2005)

Bottom Line: The overexpression of members of the ErbB tyrosine kinase receptor family has been associated with cancer progression.This colocalization requires intact FAK.In summary, distinct FAK signaling has an essential function in ErbB-induced oncogenesis and invasiveness.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Lady Davis Institute of the Sir Mortimer B. Davis Jewish General Hospital, McGill University, Montreal, Quebec H3T 1E2, Canada.

ABSTRACT
The overexpression of members of the ErbB tyrosine kinase receptor family has been associated with cancer progression. We demonstrate that focal adhesion kinase (FAK) is essential for oncogenic transformation and cell invasion that is induced by ErbB-2 and -3 receptor signaling. ErbB-2/3 overexpression in FAK-deficient cells fails to promote cell transformation and rescue chemotaxis deficiency. Restoration of FAK rescues both oncogenic transformation and invasion that is induced by ErbB-2/3 in vitro and in vivo. In contrast, the inhibition of FAK in FAK-proficient invasive cancer cells prevented cell invasion and metastasis formation. The activation of ErbB-2/3 regulates FAK phosphorylation at Tyr-397, -861, and -925. ErbB-induced oncogenic transformation correlates with the ability of FAK to restore ErbB-2/3-induced mitogen-activated protein kinase (MAPK) activation; the inhibition of MAPK prevented oncogenic transformation. In contrast, the inhibition of Src but not MAPK prevented ErbB-FAK-induced chemotaxis. In migratory cells, activated ErbB-2/3 receptors colocalize with activated FAK at cell protrusions. This colocalization requires intact FAK. In summary, distinct FAK signaling has an essential function in ErbB-induced oncogenesis and invasiveness.

Show MeSH

Related in: MedlinePlus

Restoration of FAK in FAK−/− cells rescued focal adhesions and ErbB-2 relocalization to focal protrusions. (A) FAK−/− cells cooverexpressing ErbB-2 and -3 receptors were stably transfected with wild-type FAK as described in Materials and methods. Double immunofluorescence labeling of FAK and vinculin, a marker for focal adhesion, demonstrated that FAK−/−-2/3 cells in which FAK was restored (right) exhibited many focal adhesions that were labeled for both FAK and vinculin, which is similar to normal FAK+/+-2/3 cells. (B) Double immunofluorescence of FAK and ErbB-2 revealed that the restoration of wild-type FAK in FAK−/−-2/3 cells induces the relocalization of ErbB-2 receptors from the cell membrane to fingerlike protrusions. This ErbB-2 pattern is similar to FAK+/+-2/3 cells in which FAK and ErbB-2 receptors colocalized at cell protrusions. Bar, 30 μm. (C) Expression of NH2 terminus (NT) and COOH terminus (CT) FAK in FAK−/−-2/3. Cells expressing the NT or CT were stimulated without (C) or with 20 ng/ml EGF (E) or 20 ng/ml HRG (H) for 30 min. Equal amounts of protein were immunoprecipitated with anti–FAK-A17, which targets at NT-FAK, or anti-FAK–C-20 which targets at CT-FAK. Blots were probed with antiphosphotyrosine antibody. (D and E) Immunofluorescence analysis. FAK−/−-2/3 cells expressing control, CT-FAK–GFP, or NT-FAK–GFP fusion protein (see Materials and methods). After transfection, the cells were incubated in complete medium for 24 h and were fixed and coimmunostained for FAK and vinculin (D) or FAK and ErbB-2 receptor (E). Note that focal adhesions were seen only when cells were transfected with CT-FAK but not with NT-FAK (D). In contrast, cells transfected with CT-FAK exhibited a homogeneous distribution of ErbB-2 receptors at the cell membrane, whereas transfection with NT-FAK exhibited a reduced staining of ErbB-2 throughout the cytoplasm (E). Bar, 30 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171271&req=5

fig6: Restoration of FAK in FAK−/− cells rescued focal adhesions and ErbB-2 relocalization to focal protrusions. (A) FAK−/− cells cooverexpressing ErbB-2 and -3 receptors were stably transfected with wild-type FAK as described in Materials and methods. Double immunofluorescence labeling of FAK and vinculin, a marker for focal adhesion, demonstrated that FAK−/−-2/3 cells in which FAK was restored (right) exhibited many focal adhesions that were labeled for both FAK and vinculin, which is similar to normal FAK+/+-2/3 cells. (B) Double immunofluorescence of FAK and ErbB-2 revealed that the restoration of wild-type FAK in FAK−/−-2/3 cells induces the relocalization of ErbB-2 receptors from the cell membrane to fingerlike protrusions. This ErbB-2 pattern is similar to FAK+/+-2/3 cells in which FAK and ErbB-2 receptors colocalized at cell protrusions. Bar, 30 μm. (C) Expression of NH2 terminus (NT) and COOH terminus (CT) FAK in FAK−/−-2/3. Cells expressing the NT or CT were stimulated without (C) or with 20 ng/ml EGF (E) or 20 ng/ml HRG (H) for 30 min. Equal amounts of protein were immunoprecipitated with anti–FAK-A17, which targets at NT-FAK, or anti-FAK–C-20 which targets at CT-FAK. Blots were probed with antiphosphotyrosine antibody. (D and E) Immunofluorescence analysis. FAK−/−-2/3 cells expressing control, CT-FAK–GFP, or NT-FAK–GFP fusion protein (see Materials and methods). After transfection, the cells were incubated in complete medium for 24 h and were fixed and coimmunostained for FAK and vinculin (D) or FAK and ErbB-2 receptor (E). Note that focal adhesions were seen only when cells were transfected with CT-FAK but not with NT-FAK (D). In contrast, cells transfected with CT-FAK exhibited a homogeneous distribution of ErbB-2 receptors at the cell membrane, whereas transfection with NT-FAK exhibited a reduced staining of ErbB-2 throughout the cytoplasm (E). Bar, 30 μm.

Mentions: Next, we confirmed the colocalization of FAK and ErbB-2 in FAK−/−-2/3 cells in which FAK was reconstituted by immunofluorescence. FAK−/−-2/3–FAK cells exhibited many focal adhesions that were labeled for both FAK and vinculin, which is similar to FAK+/+-2/3 cells (Fig. 6 A). The restoration of FAK in FAK−/−-2/3 cells induced the relocalization of ErbB-2 from the straight parts of the cell membrane to fingerlike protrusions in a similar pattern to that seen in FAK+/+-2/3 cells (Fig. 6 B).


FAK signaling is critical for ErbB-2/ErbB-3 receptor cooperation for oncogenic transformation and invasion.

Benlimame N, He Q, Jie S, Xiao D, Xu YJ, Loignon M, Schlaepfer DD, Alaoui-Jamali MA - J. Cell Biol. (2005)

Restoration of FAK in FAK−/− cells rescued focal adhesions and ErbB-2 relocalization to focal protrusions. (A) FAK−/− cells cooverexpressing ErbB-2 and -3 receptors were stably transfected with wild-type FAK as described in Materials and methods. Double immunofluorescence labeling of FAK and vinculin, a marker for focal adhesion, demonstrated that FAK−/−-2/3 cells in which FAK was restored (right) exhibited many focal adhesions that were labeled for both FAK and vinculin, which is similar to normal FAK+/+-2/3 cells. (B) Double immunofluorescence of FAK and ErbB-2 revealed that the restoration of wild-type FAK in FAK−/−-2/3 cells induces the relocalization of ErbB-2 receptors from the cell membrane to fingerlike protrusions. This ErbB-2 pattern is similar to FAK+/+-2/3 cells in which FAK and ErbB-2 receptors colocalized at cell protrusions. Bar, 30 μm. (C) Expression of NH2 terminus (NT) and COOH terminus (CT) FAK in FAK−/−-2/3. Cells expressing the NT or CT were stimulated without (C) or with 20 ng/ml EGF (E) or 20 ng/ml HRG (H) for 30 min. Equal amounts of protein were immunoprecipitated with anti–FAK-A17, which targets at NT-FAK, or anti-FAK–C-20 which targets at CT-FAK. Blots were probed with antiphosphotyrosine antibody. (D and E) Immunofluorescence analysis. FAK−/−-2/3 cells expressing control, CT-FAK–GFP, or NT-FAK–GFP fusion protein (see Materials and methods). After transfection, the cells were incubated in complete medium for 24 h and were fixed and coimmunostained for FAK and vinculin (D) or FAK and ErbB-2 receptor (E). Note that focal adhesions were seen only when cells were transfected with CT-FAK but not with NT-FAK (D). In contrast, cells transfected with CT-FAK exhibited a homogeneous distribution of ErbB-2 receptors at the cell membrane, whereas transfection with NT-FAK exhibited a reduced staining of ErbB-2 throughout the cytoplasm (E). Bar, 30 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171271&req=5

fig6: Restoration of FAK in FAK−/− cells rescued focal adhesions and ErbB-2 relocalization to focal protrusions. (A) FAK−/− cells cooverexpressing ErbB-2 and -3 receptors were stably transfected with wild-type FAK as described in Materials and methods. Double immunofluorescence labeling of FAK and vinculin, a marker for focal adhesion, demonstrated that FAK−/−-2/3 cells in which FAK was restored (right) exhibited many focal adhesions that were labeled for both FAK and vinculin, which is similar to normal FAK+/+-2/3 cells. (B) Double immunofluorescence of FAK and ErbB-2 revealed that the restoration of wild-type FAK in FAK−/−-2/3 cells induces the relocalization of ErbB-2 receptors from the cell membrane to fingerlike protrusions. This ErbB-2 pattern is similar to FAK+/+-2/3 cells in which FAK and ErbB-2 receptors colocalized at cell protrusions. Bar, 30 μm. (C) Expression of NH2 terminus (NT) and COOH terminus (CT) FAK in FAK−/−-2/3. Cells expressing the NT or CT were stimulated without (C) or with 20 ng/ml EGF (E) or 20 ng/ml HRG (H) for 30 min. Equal amounts of protein were immunoprecipitated with anti–FAK-A17, which targets at NT-FAK, or anti-FAK–C-20 which targets at CT-FAK. Blots were probed with antiphosphotyrosine antibody. (D and E) Immunofluorescence analysis. FAK−/−-2/3 cells expressing control, CT-FAK–GFP, or NT-FAK–GFP fusion protein (see Materials and methods). After transfection, the cells were incubated in complete medium for 24 h and were fixed and coimmunostained for FAK and vinculin (D) or FAK and ErbB-2 receptor (E). Note that focal adhesions were seen only when cells were transfected with CT-FAK but not with NT-FAK (D). In contrast, cells transfected with CT-FAK exhibited a homogeneous distribution of ErbB-2 receptors at the cell membrane, whereas transfection with NT-FAK exhibited a reduced staining of ErbB-2 throughout the cytoplasm (E). Bar, 30 μm.
Mentions: Next, we confirmed the colocalization of FAK and ErbB-2 in FAK−/−-2/3 cells in which FAK was reconstituted by immunofluorescence. FAK−/−-2/3–FAK cells exhibited many focal adhesions that were labeled for both FAK and vinculin, which is similar to FAK+/+-2/3 cells (Fig. 6 A). The restoration of FAK in FAK−/−-2/3 cells induced the relocalization of ErbB-2 from the straight parts of the cell membrane to fingerlike protrusions in a similar pattern to that seen in FAK+/+-2/3 cells (Fig. 6 B).

Bottom Line: The overexpression of members of the ErbB tyrosine kinase receptor family has been associated with cancer progression.This colocalization requires intact FAK.In summary, distinct FAK signaling has an essential function in ErbB-induced oncogenesis and invasiveness.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Lady Davis Institute of the Sir Mortimer B. Davis Jewish General Hospital, McGill University, Montreal, Quebec H3T 1E2, Canada.

ABSTRACT
The overexpression of members of the ErbB tyrosine kinase receptor family has been associated with cancer progression. We demonstrate that focal adhesion kinase (FAK) is essential for oncogenic transformation and cell invasion that is induced by ErbB-2 and -3 receptor signaling. ErbB-2/3 overexpression in FAK-deficient cells fails to promote cell transformation and rescue chemotaxis deficiency. Restoration of FAK rescues both oncogenic transformation and invasion that is induced by ErbB-2/3 in vitro and in vivo. In contrast, the inhibition of FAK in FAK-proficient invasive cancer cells prevented cell invasion and metastasis formation. The activation of ErbB-2/3 regulates FAK phosphorylation at Tyr-397, -861, and -925. ErbB-induced oncogenic transformation correlates with the ability of FAK to restore ErbB-2/3-induced mitogen-activated protein kinase (MAPK) activation; the inhibition of MAPK prevented oncogenic transformation. In contrast, the inhibition of Src but not MAPK prevented ErbB-FAK-induced chemotaxis. In migratory cells, activated ErbB-2/3 receptors colocalize with activated FAK at cell protrusions. This colocalization requires intact FAK. In summary, distinct FAK signaling has an essential function in ErbB-induced oncogenesis and invasiveness.

Show MeSH
Related in: MedlinePlus